PROJECT SUMMARY Pathogenic bacteria must assemble and secrete virulence factors to interact with host tissues and cause disease. Gram-negative bacteria have an outer membrane in addition to a cytoplasmic membrane, and must secrete virulence factors across both these barriers. The mechanisms by which this occurs can be quite complex and are not well understood. We are using the chaperone/usher (CU) pathway as a model system with which to probe mechanisms of protein secretion and virulence factor biogenesis in Gram-negative bacteria. The CU pathway is a conserved secretion system dedicated to the assembly of virulence-associated surface structures termed pili or fimbriae. Pili are hair-like polymers that typically function as adhesive organelles and have roles in colonization of surfaces, biofilm formation, interactions with host cells, and pathogenesis. The prototype structures assembled by the CU pathway are the P and type 1 pili expressed by uropathogenic Escherichia coli. E. coli is the primary causative agent of urinary tract infections, a major healthcare burden, and P and type 1 pili are critical virulence factors that mediate colonization of the kidneys and bladder, respectively. The CU pathway requires two components for secretion across the OM: a periplasmic chaperone and an integral OM protein termed the usher. The chaperone directs proper folding of subunit proteins and maintains the proteins in an assembly-competent state. The usher is a dynamic molecular machine that catalyzes the formation of subunit-subunit interactions, promotes ordered polymerization of the pilus fiber, and provides the channel for secretion of the pilus to the cell surface. The goals of this proposal are to probe the structure and function of the usher to gain an understanding of the molecular mechanisms governing pilus biogenesis across the bacterial outer membrane, and to use the CU pathway as a model system for understanding virulence factor secretion in Gram-negative bacteria. This proposal will test the hypothesis that the usher orchestrates a defined sequence of domain movements and protein-protein interactions to ensure the assembly of functional pili, and that usher molecules interact in an asymmetric manner in the OM to catalyze pilus biogenesis. The first specific aim will define mechanisms by which the usher regulates and harnesses protein-protein interactions to catalyze ordered pilus assembly. The second specific aim will determine how usher molecules interact in an asymmetric manner to catalyze pilus biogenesis with maximal efficiency. The third specific aim will reveal the structural basis of pilus biogenesis by the usher in its native state. This application will apply a multidisciplinary approach to answer questions relevant to fundamental mechanisms of protein secretion across biological membranes, virulence factor biogenesis, and the assembly of complex organelles.